Title: Enhancement of anticancer therapy by flavonoids.

The invention relates to the synergistic use of a polyphenolic flavonoid and a chemotherapeutic drug for treating an individual suffering from cancer

Cancer is the uncontrolled growth and spread of cells that may affect almost any tissue of the body. Lung, colorectal and stomach cancer are among the five most common cancers in the world for both men and women. Among men, lung and stomach cancer are the most common cancers worldwide. For women, the most common cancers are breast and cervical cancer. More than 11 million people are yearly diagnosed with cancer worldwide, of which more than 60% will die because of the cancer.

Conventional treatment comprises surgery, radiation, chemotherapy, hormonal therapy and immunotherapy. Hormone therapy, chemotherapy and immunotherapy are systemic treatments which may affect cancer cells throughout the body, while surgery and radiation are used to eliminate or eradicate local cancer cells.

Surgery involves the removal of the cancer and some tissue adjacent to the cancer. Radiation or radiotherapy, uses high-energy rays to damage or kill cancer cells by preventing them from growing and dividing. Chemotherapy involves the use of drugs, often termed chemotherapeutic drugs, to eliminate cancer cells. Chemotherapy is often used alone, or in conjunction with radiation therapy or surgery. Hormonal therapy is often used to eliminate cancer cells that are responsive to hormones such as oestrogen and androgen. Typical examples of such cancer cells are breast cells and prostate cells.

Hormonal therapy includes the use of drugs that block hormone production, the removal of organs that secrete hormones, and the use of drugs that change

the way hormones work, such as anti-hormones. Other systemic anticancer therapies are immunotherapy and targeted therapy. Immunotherapy comprises the use of interferon and other cytokines to stimulate the natural defense system to eliminate cancer cells, and also includes the use of foreign antibodies and cancer vaccines.

Conventional cancer treatments, such as chemotherapy and radiation therapy, do not distinguish between cancer cells and healthy cells. Chemotherapy aims at eliminating rapidly dividing cells in general, which is a hallmark of cancer cells. However, there are also healthy cells that can be rapidly dividing, such as blood cells and the cells lining the mouth and the gut, and which therefore may also be damaged by chemotherapy. Similarly, radiation therapy kills healthy cells that are in the path of the radiation. The damage of healthy cells may result in side effects, such as nausea, vomiting, hair loss and mouth sores. These side effects may be severe, and thereby reducing the quality of life of a particular patient. In some cases, the side effects may even prevent full treatment of a particular patient. However, many side effects can be prevented or controlled, allowing many people to normally take part in life while receiving chemotherapy.

For example, the administration of doxorubicin to patients is limited by a dose-dependent cardiotoxicity, which can lead to late side-effects resulting in severe morbidity and also mortality. Combining doxorubicin treatment with other anticancer drugs, e.g. taxanes, increases efficacy, but unfortunately also augments cardiotoxicity (Minotti et al., 2004. Pharmacol Rev 56: 185; Seidman et al., 2002. J Clin Oncol 20: 1215). Preclinical experiments showed that a polyphenols flavonoid such as 7-monohydroxyethylrutoside (monoHER) is a potential protective agent against doxorubicin-induced cardiotoxicity that has no interference with its antitumor activity (Van Acker SA et al., 1997. Clin Cancer Res 3: 1747; Van Acker et al., 2000. Clin Cancer Res 6: 1337). The

radical scavenging and iron chelation properties of monoHER are supposed to be the mechanisms of action (Haenen et al., 1993. Venoruton. Phlebology : SlO; Van Acker et al., 1993. Phlebology: S31). In a phase I study the possible side effects and pharmacokinetics of monoHER were evaluated. No side-effects were observed and the potentially cardioprotecting pharmacokinetic values for Cmax and AUC°° were achieved (Willems et al., 2006. Cancer Chemother Pharmacol 57:678).

Many patients diagnosed with cancer receive chemotherapy. For cancers that respond well to the prescribed chemotherapeutic drugs, this approach will treat the cancer effectively. However, several cancers do not respond, or only marginally respond, to the prescribed chemotherapeutic drugs. Therefore, it is an object of the present invention to enhance the chemotherapeutic effect of a chemotherapeutic drug in humans.

Therefore, in one aspect the invention provides the use of a polyphenols flavonoid in the manufacture of a medicament for the treatment of an individual suffering from cancer, whereby said flavonoid acts synergistically with a chemotherapeutic drug; and whereby said individual is a human.

Upon conducting phase II experiments, the inventors surprisingly established that a polyphenols flavonoid such as monoHER, a compound that was thusfar used to prevent cardiotoxicity induced by a chemotherapeutic drug, synergistically increases efficacy of said chemotherapeutic drug.

A chemotherapeutic drug is preferably a drug that affects rapidly dividing cells such as cancer cells. In general, such a chemotherapeutic drug acts by hampering or preventing proliferation of cells by damaging the DNA, inhibiting the synthesis or replication of DNA, or blocking the separation of

the daughter cells. Commonly used chemotherapeutic drugs can be grouped into alkylating agents such as cyclophosphamide and cisplatin, nitrosoureas such as carmustine and semustine, antimetabolites such as mercaptopurine and 5-fluorouracil, antibiotics such as anthracyclines and mitomycin-C, alkaloids such as vincristine and taxane, and hormones such as tamoxifen.

In a preferred embodiment, said chemotherapeutic drug comprises an anthracycline.

Anti-tumor antibiotics in general are known to eliminate cancer cells by preventing DNA replication and/or RNA synthesis. Anthracyclines have been reported to inhibit proliferation of cancer cells by multiple mechanisms, including but not limited to intercalating with cellular DNA, binding to cell membranes, altering ion transport, and generating oxygen radicals that induce DNA breaks. Anthracyclines, such as daunorubicin, idarubicin, doxorubicin, and epirubicin, are used to treat multiple cancer types, including breast cancer, ovarian cancer, soft tissue and osteogenic sarcomas, neuroblastoma, lymphoma, and leukemia.

The anthracycline doxorubicin (DOX) is widely used in the treatment of several solid tumors and hematological malignancies in adult and pediatric patients. Treatment with DOX is limited by a dose-dependent cardiotoxicity, which may lead to late side effects resulting in severe morbidity and mortality (1, 2). Although the 5-year survival of childhood cancer has improved from 30% to 70% in the last 40 years, the risk of death from cardiac events in these survivors is eight times higher than that in the normal population (3). Besides this, combining DOX with other anticancer drugs, e.g. taxanes and trastuzumab, increases efficacy, but unfortunately also augments cardiotoxicity (4, 5).

Although the mechanism of DOX-induced cardiotoxicity is still not fully understood, a major role has been ascribed to the induction of free radicals (6-8). The cardiomyocyte is particularly vulnerable to free radical injury because of properties such as a low antioxidant status (9, 10). Presently, the cardioprotectant dexrazoxane is the only drug with proven efficacy (11). A recent review recommended the use of dexrazoxane if the risk of cardiotoxicity is high. However, clinicians should weigh the cardioprotective effect of this agent against the risk of a potential decrease of antitumor activity for the individual patient (12). Preclinical experiments showed that the flavonoid 7- monohydroxyethylrutoside (monoHER) is also a potential protective agent against DOX-induced cardiotoxicity without interfering with its antitumor activity (13, 14). The radical scavenging and iron chelating properties of monoHER are its supposed mechanisms of action (15, 16). No serious side effects were observed in a clinical phase I study up to a dose of 1500 mg/m ² . At this dose the pharmacokinetic end-points were reached, i.e. Cmax and AUC were comparable to those obtained in mice under protecting conditions. Therefore this dose was considered feasible and safe to be evaluated in a phase II study (17). In the present invention it is preferred that the chemotherapeutic drug is doxorubicin.

In a preferred embodiment, said polyphenolic flavonoid comprises a compound according to formula 1:

formula 1 wherein: A and E form together a C-C or C=C bond, Ri, R2, R3, and R ₄ are each independently chosen from (or selected from the group consisting of): H; OH;

(ROn R" and/or O(R0n R" wherein n=0-8 and R' is independently chosen from CH2 or Q(CEb) _n ¹ wherein ^=1-4 and

R" when n=0 is independently chosen from H; an aromatic group; a Ci-Cs alkyl group; a sugar in mono, di or trimeric form or an analogue thereof; halide;

NHR ¹ "; N(R ¹¹ O ₂ ; N(R"O ₃ -halide; COOR"';

CONHR ¹ "; CON(R'")2; or CON(R'") ₃ -halide wherein R'" is independently chosen from H; an aromatic group; a

Ci-Cs alkyl group;or a sugar in mono, di or trimeric form or an analogue thereof; and when

n=l-8 is independently chosen from H; an aromatic group; a C ₁ -Cs alkyl group; a sugar in mono, di or trimeric form or an analogue thereof; NHR"'; N(R ^m ) ₂ ; N(R ^m ) ₃ -halide;;

OH; halide; COOR"';

CONHR" ¹ ; CON(R'") ₂ ; or CON(R"') ₃ -halide wherein R'" is independently chosen from H; an aromatic group; a

C ₁ -Ce alkyl group; or a sugar in mono, di or trimeric form or an analogue thereof; (R')nOR" wherein n=l-8 and

R' is independently chosen from CH ₂ or O(CH ₂ ) _n ' wherein n'=l-4 and R" is independently chosen from

H; an aromatic group; a C ₁ -Cs alkyl group; a sugar in mono, di or trimeric form or an analogue thereof; NHR'"; N(R'") ₂ ; N(R'") ₃ -halide; H; halide; COOR'";

CONHR"'; CON(R'") ₂ or CON(R'") ₃ -halide wherein R'" is independently chosen from H; an aromatic group; a C ₁ -Cs alkyl group; or a sugar in mono, di or trimeric form or an analogue thereof; wherein n, n' and q are integers and wherein the number range indication letter=(number l)-(number 2) means that the letter can be any of the integers from and including number 1 to and including number 2. The alternatives for each R ₁ ; R _2; R ₃ ,- R ₄ ; R';R" and R'" can be independently chosen.

Polyphenolic flavonoids comprise a class of naturally occurring benzo-γ- pyron derivatives that are ubiquitous in photosynthesizing cells and provide colour, flavour, anti-fungal and antibacterial activity. Sources of natural flavonoids comprise dark chocolate, strawberries, blueberries, cinnamon, pecans, walnuts, grapes, lemons, and cabbage. Besides their relevance in plants, they are pharmacologically important as they comprise iron-chelating and radical- scavenging properties, which may contribute to the antioxidant activity of flavonoids. Fully synthetic and semi-synthetic compounds have recently become available.

Natural polyphenolic flavonoids comprise flavon(ol)es such as quercitin and rutin, flavonon(ol)es such as naringin and hesperetin, flavones such as luteolin, flavanoles such as catechin, chalcones such as phloretin, anthocyanidins such as cyanidin and isoflavones such as genistein.

In yet a further preferred embodiment, said polyphenolic flavonoid comprises a compound according to formula 1 (supra) with the proviso that said compound is not a compound wherein R ₁ = OH, and and E together form a C=C bond. Preferably said compound is not quercitin.

Semi-synthetic polyphenolic flavonoids comprise hydroxyethylated- derivatives such as hydroxyethylrutosides among which 7- monohydroxyethylrutoside (mono-HER). In a further preferred embodiment, said polyphenolic flavonoid comprises monohydroxyethylrutoside.

Synthetic polyphenolic flavonoids, and novel methods for their production, have been described in WO/2001/21608, which is incorporated herein by reference.

Semisynthetic and synthetic polyphenolic flavonoids have advantages over natural polyphenolic flavonoids. It was surprisingly found by the present inventors that the semisynthetic polyphenolic flavonoid monoHER and compounds of formula 1 that have the same or similar R ₁ , R2, R3 or R4 groups are with respect to their anti-tumor activity more synergistic with a chemotherapeutic drug. Thus preferably said polyphenolic flavonoid of formula 1 is a synthetic or semi-synthetic flavonoid. Preferably said flavonoid of formula 1 is not a naturally occurring flavonoid. In a preferred embodiment said compound of formula I is a hydroxy-ethylated or hy droxy-me thy late d flavonoid. In a particularly preferred embodiment said polyphenolic flavonoid comprises monoHER.

Flavonoids such as monoHER can protect against oxidative stress by efficiently scavenging free radicals. During this process, the antioxidant is converted into a reactive oxidation product that is converted back into its parent compound by ascorbate or captured by GSH to form a glutathionyl adduct. Flavonoids can form harmful oxidation products that might induce cellular damage. It was found that distinct flavonoids give rise to different oxidation products. For example, monoHER produces less damaging oxidation products compared to quercetin, suggesting that monoHER acts less as a pro- oxidant compared to quercetin and therefore, will produce less side effects. Furthermore, the oxidation products of specific flavonoids, such as for example monoHER, can be readily recycled by, for example, ascorbate. For monoHER, it was found that the reaction with ascorbate takes place approximately 3 times faster than with GSH, further substantiating an advantage of monoHER over quercetin in respect of functioning as a pro-oxidant.

Chemotherapeutic drugs are used for the treatment of a diverse set of cancer types. Cancers can be classified either according to the kind of fluid or

tissue from which they originate, or according to the location in the body where they first developed. Cancer types that might be treated by a synergistic effect of a polyp henolic flavonoid and a chemotherapeutic drug according to the invention include, but are not limited to, a carcinoma, which is a cancer of epithelial tissue that covers or lines surfaces of organs, glands, or body structures and which account for 80 percent to 90 percent of all cancer cases; a sarcoma, which is a cancer growing from connective tissues, such as cartilage, fat, muscle, tendons, and bones; a lymphoma, which is a cancer type that originates in the nodes or glands of the lymphatic system; a leukemia, which is a cancer of the bone marrow; or a myeloma, which is a cancer that grows in the plasma cells of bone marrow.

In this aspect, the invention preferably provides the use of a polyphenolic flavonoid in the manufacture of a medicament for the treatment of an individual suffering from cancer, wherein said cancer comprises a soft tissue sarcoma.

The inventors have surprisingly found that a polyphenolic flavonoid acts synergistically with a chemotherapeutic drug for the treatment of soft tissue sarcomas.

The term soft tissue refers to tissues that connect, support, or surround other structures and organs of the body. Soft tissue includes muscles, tendons, fibrous tissues, fat, blood vessels, nerves, and synovial tissues. Soft tissue sarcomas in general share clinical and microscopic characteristics that are different from osteosarcomas and are treated differently from said osteosarcomas.

Some tumors of the soft tissue are termed benign or non-cancerous. These tumors do not spread and are rarely life-threatening. However, some

benign tumors can cause symptoms due to interference with normal body functions.

Sarcomas can invade surrounding tissue and can metastasize to other organs of the body, forming metastases. The cells of these metastases or secondary tumors are similar to those of the primary cancer.

Therefore, the invention further provides the use of a polyphenolic flavonoid in the manufacture of a medicament for the treatment of an individual suffering from a sarcoma, comprising the treatment of an individual suffering from metastases of said sarcoma.

In a preferred embodiment, said sarcoma is selected from the group comprising alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, mesenchymoma, clear cell sarcoma, chondrosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma , synovial sarcoma,and peripheral nerve sheet tumor.

In yet a further preferred embodiment, said sarcoma is selected from the group comprising fibrous histiocytoma and peripheral nerve sheet tumor. Polyphenolic flavonoids have been used in preclinical experiments to test their cardioprotective effects upon treatment of the animals with an anthracycline such as doxorubicin (van Acker et al., Clin Cancer Res. 1997. 3(10): 1747). From a first set of experiments, it was concluded that polyphenolic flavonoids such as monoHER showed a dose-dependent cardioprotection against doxorubicin- induced chronic cardiotoxicity and did not influence the antitumor activity of doxorubicin in vitro or in vivo. However, subsequent experiments indicated that this cardioprotective effect was not prolonged during a half year

observation period, suggesting that the dose and frequency of administration of polyp henolic flavonoids have an effect.

Polyphenolic flavonoids can be administered into the body by several routes. They may be taken orally, injected intravenously, intramuscularly, intrathecally, or subcutaneously, or introduced by insertion into the rectum or vagina. When injected intravenously, said polyphenolic flavonoid is immediately delivered to the bloodstream and tends to take effect more quickly than when given by any other route.

Polyphenolic flavonoids can also be given by intravenous, intramuscular, intrathecal, or subcutaneous administration. Parameters that are preferably controlled during administration of the polyphenolic flavonoid are dose and time.

Therefore, in another embodiment, said polyphenolic flavonoid is administered to said individual by intravenous infusion at a dose of between 100 and 3000 mg/m ² .

In a preferred embodiment, said polyphenolic flavonoid is administered to said individual by intravenous infusion at a dose of 1500 mg/m ² .

In a further preferred embodiment, said polyphenolic flavonoid is administered to said individual by infusion of a total volume of between about 1 and about 1000 ml, more preferred between about 2 and about 500 ml, more preferred between about 5 and about 250 ml. The limitations provided may prevent fractional changes in blood volume and may prevent further complications.

A polyp henolic flavonoid of the invention can be administered to the individual during the administration of said chemotherapeutic drug, after administering said chemotherapeutic drug, or prior to administering said chemotherapeutic drug. If the polyp henolic flavonoid is administered simultaneously with said chemotherapeutic drug, said polyphenolic flavonoid and said chemotherapeutic drug may be comprised in one pharmaceutical composition, or in more than one pharmaceutical composition, further comprising an acceptable carrier. A polyphenolic flavonoid of the invention is preferably administered prior to administration of the chemotherapeutic drug.

It is possible to administer the polyphenolic flavonoid or the chemotherapeutic drug in a very short time for instance via intravenous injection or over an extended period of time. Preferably, however the polyphenolic flavonoid or the chemotherapeutic drug is administered over a time period between 0 and 24 hours, preferably over a period of 0-12 hours and more preferably over a period of 0-6 hours. A practical time frame is between 0 and 2 hours.

While it is possible to administer the polyphenolic flavonoid and the chemotherapeutic drug simultaneously, it is preferred that the individual is first provided with the polyphenolic flavonoid and subsequently with the chemotherapeutic drug. In this embodiment it is possible to start administration of the chemotherapeutic drug before completion of the administration of said polyphenolic flavonoid of the invention. In another embodiment it is preferred that the polyphenolic flavonoid administration is completed before the administration of the chemotherapeutic drug is started. In a preferred embodiment, the time interval between the end of administration of the polyphenolic flavonoid and the start of administration of the chemotherapeutic drug is between 0 and 24 hours, more preferably

between 0 and 12 hours, more preferred between 0 and 6 hours, even more preferred between 0 and 2 hours.

When the chemotherapeutic drug administration is started prior to the start of administration of the polyp henolic flavonoid of the invention, it is preferred that polyphenolic flavonoid administration is started as early as possible after initiation of the administration of said chemotherapeutic drug. Preferably said polyphenolic flavonoid administration is started between 0-24 hours of initiation of the administration of said chemotherapeutic drug. More preferred between 0-12, more preferred between 0-6 and even more preferred between 0-2 hours of initiation of the administration of said chemotherapeutic drug.

In yet another aspect, the invention provides a method for treating an individual suffering from cancer, comprising administering to said individual a medicament comprising a polyphenolic flavonoid and a chemotherapeutic drug, whereby said flavonoid acts synergistically with said chemotherapeutic drug; and whereby said individual is a human.

In one embodiment, said chemotherapeutic drug comprises an anthracycline, preferably said anthracycline is doxorubicin.

In a further embodiment, said polyphenolic flavonoid comprises a compound according to formula 1 (supra).

In yet a further embodiment, the invention provides a method for treating an individual suffering from cancer, comprising administering to said individual a medicament comprising a polyphenolic flavonoid and a medicament comprising a chemotherapeutic drug, wherein said medicament

comprising a polyphenolic flavonoid is administered prior to administering said medicament comprising a chemotherapeutic drug.

In another aspect, the invention provides the use of a polyphenolic flavonoid for the manufacture of a medicament to enhance the chemotherapeutic effect of a chemotherapeutic drug in human.

In yet another aspect, the invention provides a pharmaceutical composition, comprising a polyphenolic flavonoid and an acceptable carrier.

The invention further provides the use of a polyphenolic flavonoid according to the invention for enhancement of the chemotherapeutic effect of a chemotherapeutic drug, preferably anthracycline, more preferably doxorubicin, whereby it is preferred that said use of a polyphenolic flavonoid is for treatment of a human individual suffering from soft tissue sarcoma.

It was found by the present investigators that polyphenolic flavonoids, such as monoHER, potentiate the chemotherapeutic effect of a chemotherapeutic drug, preferably an anthracycline such as doxorubicin.

It is further preferred that said polyphenolic flavonoid such as monoHER is administered to said individual by intravenous infusion at a dose of between 100 and 3000 mg/m2, more preferred between 100 and 2000 mg/m2, more preferred between 500 and 2000 mg/m2, more preferred between 1000 and 2000 mg/m2, most preferred about 1500 mg/m2. It is furthermore preferred that said polyphenolic flavonoid is administered to said individual prior to administering said chemotherapeutic drug. In this embodiment it is possible to start administration of the chemotherapeutic drug before completion of the administration of said polyphenolic flavonoid

In a further preferred embodiment, said polyphenolic flavonoid is administered to said individual by infusion of a total volume of between about 1 and about 1000 ml, more preferred between about 2 and about 500 ml, more preferred between about 5 and about 250 ml. The limitations provided may prevent fractional changes in blood volume and may prevent further complications.

Brief description of the drawings

Figure 1. Heart tissue from patient #3 after 300 mg/m ² DOX. Evaluation by electron microscopy demonstrated an abundant presence of microvacuoles in the cardiomyocytes.

Figure 2. Heart tissue from patient #3 after 480 mg/m ² DOX. In addition to the vacuolization, a loss of myofibrils is observed with electron microscopy.

Figure 3. Effect of co-treatment with monoHER on growth inhibition induced by doxorubicin in WLS- 160 cells (3A); SKLMS-I cells (3B), and SK-UT-I cells (3C).

Figure 4. Effect of pre-treatment with monoHER for 1 hour (4A), 6 hours (4B) and 24 hours (4C) on growth inhibition induced by doxorubicin in WLS- 160 cells.

Figure 5. Effect of different doxorubicin concentrations for different time points on NF-kB activation in WLS-160 cells (A). Effect of pre-treatment with monoHER on doxorubicin-induced NF-kB activation.

Figure 6. UVWIS spectra (absorbance vs. wavelength (nm)) of 50 μM monoHER (A); 50 uM monoHER + 1.6 nM HRP + 33 uM H2O2 (B); 50 uM monoHER + 1.6 nM HRP + 33 uM H2O2 + 40 uM ascorbate (C); 50 uM monoHER + 1.6 nM HRP + 33 uM H2O2 + 40 uM GSH (D); 50 uM monoHER + 1.6 nM HRP + 33 uM H2O2 + 40 uM GSH + 40 uM ascorbate (E).

Figure 7. HPLC analysis of 50 μM monoHER (A); 50 uM monoHER + 1.6 nM HRP + 33 uM H2O2 (B); 50 uM monoHER + 1.6 nM HRP + 33 uM H2O2 + 40 uM ascorbate (C); 50 uM monoHER + 1.6 nM HRP + 33 uM H2O2 + 40 uM

GSH (D); 50 uM monoHER + 1.6 nM HRP + 33 uM H2O2 + 40 uM GSH + 40 uM ascorbate (E).

Figure 8. Decrease per minute in monoHER concentration, determined spectrophotometrically, in the incubation mixtures containing monoHER (50 μM), HRP (1.6 nM) and H2O2 (33 μM) in the presence of either ascorbate (40 μM), GSH (40 μM) or both ascorbate (40 μM)and GSH (40 μM). All measurements were carried out in triplicate and are expressed as means ± SD.

Figure 9. Decrease per minute in monoHER and ascorbate concentration determined spectrophotometrically in the incubation mixtures containing monoHER (50 μM), HRP (1.6 nM), H2O2 (33 μM) and ascorbate (0, 25, 40, or 100 μM). Ascorbate concentrations of 25, 40, and 100 μM decrease the HRP activity for respectively 55, 60 and 94 %. All measurements were carried out in triplicate and are expressed as means ± SD.

Figure 10. Average rate of GSH-adduct formation compared to the average rate of ascorbate consumption in the incubation mixtures containing monoHER (50 μM), HRP (1.6 nM) and H2O2 (33 μM) in the presence of both GSH (40, 50 or 5000 μM) and ascorbate (40 μM). Ascorbate reacts approximately 3 times faster than GSH with oxidized monoHER when both compounds are presented in the same concentration.

Figure 11. Aromatic parts of the IH NMR spectra of the parent compound monoHER (A) and the metabolite formed in the incubation of monoHER with HRP in the presence of GSH (B).

Figure 12. Overview of the oxidation products of monoHER.

Examples

Example 1

In this example, we evaluated the cardioprotective properties of monoHER given as a 10 minute i.v infusion at a dose of 1500 mg/m ² in patients with metastatic cancer treated with DOX. For the early detection of DOX-induced cardiotoxicity endomyocardial biopies were taken, because of its high sensitivity and high specificity (18).

PATIENTS AND METHODS Patient selection

Patients with metastatic solid tumors were entered when they received a DOX-based chemotherapy regimen with a dosage of DOX > 50 mg/m ² / cycle and an infusion duration < 1 hour. Patients had a WHO performance status of < 2 and a life expectancy of more than 3 months. They also had adequate organ functions as defined by a creatinine < 140 μmol/1 or a creatinine clearance > 60 ml/min, serum bilirubin < 26 μmol/1, alkaline phosphatase, ASAT and ALAT < 2.5x the upper limit of normal (ULN), unless related to liver metastases, in which case < 5x ULN was allowed. The left ventricular ejection fraction (LVEF) was > 50%. Patients were excluded if they had received prior anthracyclines, had prior or actual cardiovascular disease or had prior radiotherapy to the mediastinum.

All patients gave written informed consent and the protocol was approved by the medical ethical review committee of the VU University Medical Center (VUMC). Patients were enrolled between September 2003 and March 2006. Treatment

7-Monohydroxyethylrutoside (monoHER) was provided by Novartis Consumer Health (Nyon, Switzerland). The drug was formulated by the Department of Pharmacy, VUMC, Amsterdam and dissolved as described before (17).

Formulated doxorubicin (Doxorubicin hydrochloride, 2 mg/ml) was obtained from Pharmachemie B.V. (Haarlem, the Netherlands). MonoHER was administered as a 10 min intravenous infusion at a dose of 1500 mg/m ² 60 min before every DOX administration. If cardiotoxicity would be observed in the first three evaluable patients, administration according to this dosing scheme would be changed and the following patients would receive DOX infusion either immediately after monoHER (because in plasma and heart Cmax of monoHER is obtained immediately after the end of infusion (17, 19)) or with an interval of 2 hours (to give monoHER the opportunity to convert into an active metabolite, if any). Patient evaluation

Before starting the study patients were evaluated by a full blood count, serum biochemistry including liver function tests, lactate dehydrogenase (LDH) and cholesterol. Risk factors for cardiovascular disease were also evaluated. An LVEF and an ECG were performed before entry into the study.

Every subsequent administration of monoHER and DOX was preceded by a full blood count, liver enzymes, serum creatinine and a routine 12-lead ECG. A complete blood count was also done 10 days after the chemotherapy. After a cumulative dose of 300 mg/m ² DOX an endomyocardial biopsy was performed and measurement of the LVEF was repeated. The latter was also done at least 3 weeks after the last dose of DOX and a biopsy was repeated if possible. For logistic reasons, the biopsy of patient #8 was done after a cumulative dose of 375 mg/m ² DOX. Endomyocardial biopsy During a left heart catheterization, a 104 cm, 7 french biopsy forceps was used to obtain tissue from the left ventricle. Three to four specimens 0.5 to 1 mm in diameter were obtained. The specimens were fixed in 4% buffered formaldehyde and prepared for electron microscopy. Histological analysis and biopsy scores

After fixation in 4% buffered formaldehyde, the heart tissue was post fixed in 1% osmium tetroxide. The tissue was then dehydrated through a graded series of ethanol solutions of 70-95% and embedded in JB-4 Plus resin. Thereafter, 0.5—3.0 μm thick sections were cut with a glass knife. These semithin sections were processed for electron microscopy. Cardiomyocytes with more than 2 microvacuoles, macrovacuoles and/or loss of myofibrils were counted as deviant. The morphological grade determined from the specimens examined by electron microscopy was scored on a 6-point scale previously described by Billingham and Bristow (20, 21): in grade 0 cells are normal; in grade 1, 1.5, 2 and 2.5 deviant cells are < 5%, 5-15%, 16 - 25 % and 26 - 35%, respectively; in grade 3 cell damage is present in more than 35% of cells. Off-study criteria

Patients went off study in case of progressive disease, a serious cardiac event, or other events that precluded further treatment. Criteria described by Shapiro et al. (22) were used for diagnosis of cardiac events. Episodes of cardiac dysfunction were characterized according to the New York Heart Association functional classification (23). Assessment of tumor response Assessment of tumor response was done every 2-3 cycles by CT-scan, using standard ECOG criteria (24). Statistical analysis

This trial was an open-labelled, controlled study. We compared the data of our patients with those of 14 patients from the study of Torti et al. (25) treated with a cumulative dose of DOX between 200-300 mg/m ² alone using the Chi- Square test. Our hypothesis was that adding monoHER to DOX would eliminate its cardiotoxicity up to a cumulative dose of at least 300 mg/m ² , which was the upper limit of the dose interval of DOX from Torti's patients. In order to achieve a power of 80%, 11 patients would be required. This sample size was obtained when applying the Chi-Square test with significance level 0.05 and assuming a response rate (i.e. no cardiac damage) in the experimental

arm of 80%. If at least 5 patients show DOX-induced damage, statistical significance cannot be achieved anymore and the study should be stopped.

RESULTS

Eight patients meeting the inclusion criteria were enrolled (Table 1). During the analysis of risk factors, it appeared that patient no. 6 was treated for hyperhomocysteinemia. Two other patients had an elevated body mass index of 29.1 (#1), indicating overweight, and 30.1(#7), indicating obesity, respectively. Patient no. 2 had hyperlipidemia with an elevated concentration of low-density lipoproteins (LDL) of 6.3 mmol/1 (normal values < 5.0). None of the other patients had risk factors for cardiovascular disease. All patients were classified as NYHA class I and had a performance status (WHO) between 0 —2. Five patients received a cumulative dose > 300 mg/m2 DOX and underwent a biopsy. In the other three patients DOX was discontinued before this dose, due to progressive disease.

Treatment with DOX preceded by monoHER

During monoHER infusion two patients reported adverse events. One patient described a sensation of fullness in his stomach which developed during infusion of monoHER and which disappeared soon after the end of the infusion. The other patient experienced itching in the skin of the neck, starting during the infusion of monoHER and disappearing rapidly after the infusion. Both patients experienced the events during each cycle. A causal relationship cannot be excluded. The other 6 patients did not experience adverse events. None of the patients had a delay in receiving subsequent cycles of chemotherapy. As expected, all patients developed leucopenia due to chemotherapy that recovered before the start of the next cycle. No disturbances of liver enzymes and serum creatinine were noted. Biopsy scores

The endomyocardial biopsy scores are shown in Table 2. After 300 mg/m ² the first three evaluable patients showed abnormalities consistent with DOX- induced cardiotoxicity i.e. the presence of microvacuoles dominated as is shown in Figure 1. In patient #3 and #4 microvacuoles were present in 52 and 20% of the cardiomyocytes, respectively. This was also observed in patient #6, where 33% of the cardiomyocytes had > 2 small vacuoles per cardiomyocyte. Because each of the first 3 evaluable patients showed DOX-induced damage, it was decided to stop this dosing scheme. The time-interval between monoHER and DOX administration was changed and patient #7 received DOX immediately after monoHER. In the biopsy of this patient microvacuolization was observed in 60% of the cardiomyocytes. After this result again the interval was changed and patient #8 received DOX 2 h after monoHER administration. In this patient also microvacuolization was detected in 49% of the cardiomyocytes. After these results, the study had to be considered negative, because no cardioprotective effect of monoHER was observed in the five patients.

Second biopsies were also taken after 450-480 mg/m ² of DOX in two patients (Table 3). The score of patient #4 had increased from 2.0 to 2.5, whereas the score of patient #3 remained 3. Striking was the increase in loss of myofibrils in the cardiomyocytes in addition to the microvacuoles observed after 300 mg/m ² (Fig 2).

Ten months after his last cycle of DOX, patient #4 underwent a third biopsy. This time the cardiomyocytes showed recuperation of myofibrils, whereas the number of abnormal cardiac cells (microvacuolization) was less than that in the first two biopsies (score 2). No complications occurred during the 7 biopsy procedures in the first four patients. However, the last patient (#8) developed a small pericardial effusion after the biopsy. This was preceded by chest pain and transient supraventricular rhythm disturbances. These sequelae disappeared within a few days and after a week she recovered and received the sixth cycle of DOX. Monitoring and evaluation of cardiac function

In all patients the ECG remained unchanged during therapy and no cardiac dysfunction occurred.

The LVEF score of the 5 patients who received at least 300 mg/m ² DOX are shown in Table 2. All five patients started with an LVEF > 50%. After 300-375 mg/m ² DOX, the LVEF decreased in 4/5 patients. This decrease was not related with the biopsy score or the time interval between monoHER and DOX infusions. Unfortunately, no information on the LVEF was available for patient #6. Two patients received higher cumulative doses of DOX (#3 and #4). After 480 mg/m2 DOX, patient #3 had a decrease in LVEF to 53%, which is a decline of 25% compared to the ejection fraction before start of the study (71%). The patient had no symptoms of cardiac failure and even practiced fitness every day. Ten months after his last cycle of chemotherapy the LVEF remained stable and he remained without cardiac symptoms. The LVEF of patient #4 showed an initial drop from 72% before starting chemotherapy to 60% after 300 mg/m2 DOX (decline of >15%). This value remained stable up to 450 mg/m ² of DOX and at 10 month follow-up, the patient still had an excellent physical condition. Evaluation of tumor response Response to the chemotherapy was remarkable in the 4 patients with metastatic soft tissue sarcoma (STS). Three of them developed a partial remission as observed on the CT scan (Table 1). In two of these patients (#4 and #7) partial remission was maintained up to the present, i.e., 30 and 16 months after the start of chemotherapy, respectively. The other patient (#3) had progressive disease after a partial remission of nine months duration. The fourth patient (#8) achieved stable disease for at least 7 months, while continuing therapy with DOX up to a cumulative dose of 495 mg/m ² .

DISCUSSION

Based on the promising results with monoHER observed in preclinical experiments, we performed the present phase II study in patients with metastatic cancer. The cardioprotective effect of monoHER on DOX-induced cardiotoxicity was evaluated by endomyocardial biopsy. However, the results indicated that the preclinical observations were not translated into protection against DOX-induced heart damage in humans. The golden standard for early detection of DOX-induced cardiotoxicity is the endomyocardial biopsy, because of its high sensitivity and high specificity (25). Currently, the most common method used to detect DOX-induced cardiac damage is the evaluation of the LVEF, but usually at later stages (26, 27). For detection of cardiotoxicity at an earlier stage, the use of biochemical markers such as atrial and brain natriuretic peptides, endothelin-1 as well as cardiac troponin-T and -I have been investigated (26, 28, 29). However, large-scale studies addressing whether these biomarkers following DOX treatment will be predictive for the development of late onset heart failure, are lacking. Therefore, despite its invasiveness, we chose the endomyocardial biopsy for this evaluation.

Data of Billingham et al. (20) showed that anthracycline -induced myocardial damage occurred in nearly all patients treated with cumulative DOX doses of 240 mg/m ² . Therefore we could expect detectable heart damage after a cumulative dose of 300 mg/m ² , which would allow the registration of protection by monoHER. However, all 5 patients undergoing the biopsy procedure after a median cumulative dose of 300 mg/m2 showed DOX-induced cardiac damage with a mean biopsy score of 2.7 according to Billingham (20). This score was independent of the time interval between monoHER and DOX infusion and much higher than the biopsy score of 1.4, which is expected after a cumulative dose of 300 mg/m ² DOX according to the linear regression analysis of Torti et

al, (25). In our patients mainly one of the three morphological changes, i.e. small vacuoles of varying size were observed after 300 mg/m ² DOX, whereas in Torti's study also partial or total myofibrillar loss was observed (25). However, in Torti's study it is not clear after which dose this occurred. In addition to microvacuolization, loss of myofibrils was demonstrated in two patients after higher cumulative doses of DOX. This suggests that the occurrence of microvacoles as such is a marker of cytotoxicity of DOX treatment. Billingham et al. (30) described that these microvacuoles, detected by electron microscopy, appear early as a swelling of the sarcoplasmic reticulum, which eventually coalesce to form large spaces in the cytoplasm (macrovacuoles). When, or if microvacuoles always become macrovacuoles is not clear from literature. Thus, instead of protection, our patients had higher biopsy scores than the historical controls. However, it should be noted that the data of Torti et al. were obtained after a cumulative dose of 200-300 mg/m ² with biopsies from the right ventricle (25), whereas biopsies from our patients were obtained after a cumulative dose of 300 mg/m ² from the left ventricle. Previously, it was shown that ultrastructural abnormalities of myocytes were more pronounced in biopsy specimens from the left ventricle than those from the right ventricle (31). In addition to this, there is a considerable variability in patient sensitivity to the cardiotoxic effects of DOX (32, 33). This data may explain a possible overestimation of the toxic effect of DOX on the heart tissue in our patients. Although heart failure is directly related to the degree of myocyte damage (34), none of our patients developed heart failure, although the LVEF dropped by 16% in two patients, whereas it decreased in two other patients by only 4-6% (in comparison to the initial LVEF).

In the third biopsy procedure of patient #4, the score of the cardiac tissue had improved. A recuperation of myofibrils in the cardiomyocytes was observed and the microvacuolization was less. These findings are in agreement with a previous study reporting that some improvement of the histological damage

may occur (35). This is in contrast with other results, which indicated that DOX-induced cardiotoxity is an ongoing progressive process (20). The contrasting effects of monoHER found in animal and human studies may be attributed to differences in metabolism between the species. Therefore, patient #8 was treated with a 2 hour interval. However, this interval change did not reduce DOX-induced cardiac damage. In addition, no metabolites of monoHER have been detected until now. On the other hand, it cannot be excluded that during scavenging of highly reactive species in humans, the antioxidant monoHER is converted into a reactive oxidation product, which like the oxidation product of quercetin maybe prone to form adducts with thiol groups from glutathione and proteins (36). Depletion of glutathione may in addition to the low antioxidant status of the cardiac myocyte (37) reduce cardioprotection, while monoHER-protein adducts may cause additional toxicity (38). Another unexpected observation in our study was that 3 of the four patients with STS had objective remissions, while the fourth patient had stable disease. Normally, objective responses on DOX in STS patients without prior chemotherapy have been reported to be approximately 25% (39). Although our observation was done in a very limited number of patients, our result is much better than expected, because the chance of observing an objective response in 4 consecutive STS patients treated with DOX is 0.4%. Thus, monoHER enhances the antitumour activity of DOX in STS.

As a consequence of the above-mentioned aspects, there may be a dose- depending transition in the effect of monoHER i.e. a high dose (>1500 mg/m ² ) for obtaining a potentiating effect of the antitumour effect for at least soft tissue sarcomas and a low dose (somewhere below 1500 mg/m ² ) for obtaining cardioprotection. These aspects have to be elucidated further in the near future. It may be concluded that monoHER at a dose of 1500 mg/m ² did not protect against DOX-induced cardiotoxicity in patients with metastatic

disease. Further preclinical and clinical investigations seem to be warranted to investigate the dose-depending transitional effect of monoHER.

Example 2

MATERIALS AND METHODS Cell culture Human soft tissue sarcoma cell lines: WLS-160, SK-UT-I and SKLMS-I. WLS-160: liposarcoma SK-UT-I: leiomyosarcoma SKLMS-I: leiomyosarcoma

Cell growth

Exponentially growing cells were harvested and plated as single-cell suspensions in 96-well flat-bottomed microtiter plates. Cells were seeded in triplicate in 100 μl of medium at a density of 4000 cells/well After 24 h (day 1), 100 μl of medium containing the test compound monoHER was added. After 1, 6 or 24 h incubation the medium containing the test compound was removed and 200 μl of doxorubicin or medium was added. The total exposure time to the drugs was 72 h. At the end of the drug exposure period (day 4), growth- inhibitory effects were evaluated with the standard sulforhodamine B test. Briefly, microculture proteins were precipitated by addition of cold trichloroacetic acid (TCA) on the top of the growth medium and incubated for 1 h at 4 ⁰ C. The fixed cells were washed with tap water, air dried and subsequently stained with SRB; plates were washed with 1 % acetic acid, air dried and the bound protein stain was solubilised in Tris buffer. Optical density (OD) was read at 540 nm using an automated spectrophotometric microplate reader. The results were expressed as the IC50, which is the concentration of the drug giving a 50% inhibition of cell growth of treated cells when compared to the growth of control cells. Doxorubicin growth inhibition curves were plotted, and IC50 values were calculated in the presence and absence of the flavonoid.

NFkB assay

Exponentially growing cells were harvested and plated as single-cell suspensions in 6-well flat-bottomed microtiter plates. Cells were seeded in 2 ml of medium at a density of 500 000 cells/well. After 24 h, 1 ml of medium containing the test compound monoHER was added. After 1, 6 or 24 h incubation the medium containing the test compound was removed and 2 ml of doxorubicin or medium was added. After the incubation nuclear extracts were prepared. The nuclear proteins were immediately collected and stored at -80 ⁰ C until analysis. The protein concentration was determined according to the method of Bradford (BioRad, Veenendaal, The Netherlands). NF-κB activity was determined using the TransAM NF-κB p50 Activation Assay (Active Motif, Rixensart, Belgium). Results are expressed as [NF-kB] μg HeIa equivalent /ug protein. The HeIa nuclear extracts are provided a positive control for NFkB activation.

RESULTS

Effect of monoHER on growth inhibition induced by doxorubicin.

We assessed the effect of pretreatment and cotreatment of a combination of monoHER and doxorubicin on cell growth by the SRB test.

1. Cotreatment.

WLS- 160, SK-UT-I and SKLMS-I cells were treated for 72 h with either doxorubicin alone or with a combination of monoHER and doxorubicin.

DOX concentrations ranged from 0.0001 μM to 10 μM monoHER concentrations used were 50, 100, 200, 400, 800 and 1600 μM. As can be seen in Figure 3, when the cells were treated simultaneously with monoHER and doxorubicin, monoHER protects against the cell death induced by doxorubicin in a concentration dependent manner.

2. Pre treatment.

WLS- 160 cells were pretreated with monoHER (1, 6 or 24 h) before the treatment with doxorubicin. monoHER concentrations used were 50 and 800 μM

DOX concentrations used were between 0.001 μM and 10 μM.

As can be seen in Figure 4, when cells were pretreated with monoHER before the addition of doxorubicin, monoHER potentiates the growth inhibition induced by doxorubicin. This effect is not concentration dependent. The lowest monoHER concentration induces the most potentiation.

3. Effect of monoHER on the doxorubicin induced NFkB activation.

We analyzed whether doxorubicin induces NFkB activation in the WLS- 160 cell line and whether monoHER can prevent this doxorubicin induced NFkB activation.

A. Doxorubicin treatment

WLS- 160 cells were treated with different doxorubicin concentrations for different time points to determine the maximal NFkB activation induced by doxorubicin.

DOX concentrations used: 0.01, 0.1, 1 and 10 μM. Time points: 1.5, 3, 6 and 24 h.

As can be seen in Figure 5A, incubation of the cells for 6 hour with 10 μM doxorubicin induced the highest NFkB activation. This condition was used to determine the effect of monoHER preincubation on the doxorubicin-induced NFkB activation.

B. Preincubation with monoHER before treatment with doxorubicin.

WLS- 160 cells were pretreated with different monoHER concentrations before the treatment with DOX to determine the effect of monoHER on the doxorubicin-induced NFkB activation.

monoHER was used at 25, 50, 200 or 800 μM for 1 hour. DOX was used at 10 μM for 6 hours.

It was found that pretreatment of the cells with monoHER before the treatment with doxorubicin prevents the DOX-induced NFkB activation (Figure 5B).

Example 3.

MATERIALS AND METHODS

Chemicals

7-Monohydroxyethylrutoside (monoHER) was kindly provided by Novartis

Consumer Health (Nyon, Switzerland). Stock solutions of the drug were freshly prepared in a methanol/25 mM phosphate buffer mixture, pH 3.33 (4:1 v/v). Hydrogen peroxide (H2O2), horseradish peroxidase (HRP), L-ascorbic acid

(vitamin C) and reduced glutathione (GSH) were purchased from Sigma (St.

Louis, USA). Stock solutions of these chemicals were freshly prepared in a 145 mM phosphate buffer, pH 7.4.

Spectrophotometric analysis

To spectrophotometrically analyze the oxidation of monoHER, 50 μM monoHER was incubated for 5 minutes at 37 ⁰ C together with 1.6 nM HRP and 33 μM H2O2 in a 145 mM phosphate buffer. The formation of the monoHER-GSH adduct was achieved by oxidizing 50 μM monoHER in the presence of 40 μM GSH. To investigate the influence of ascorbate on the

oxidation of monoHER and on the formation of the monoHER-GSH adduct, 40 μM ascorbate was added to the incubation mixtures. All absorption spectra were recorded from 220 nm to 500 nm with a scan speed of 960 nm/min, using quartz cuvettes. The UVATIS scans were started 30 s or 300 s after the addition of HRP. MonoHER consumption was determined at 355 nm, ascorbate consumption at 270 nm.

High-Performance Liquid Chromatography (HPLC) analysis

In order to validate the results obtained spectrophotometrically, the different incubation mixtures were injected on the HPLC system 5 minutes after the addition of HRP.

HPLC was performed using a Supelcosil LC 318 column (5 μm, 25 cm x 4.6 mm) (Supelco, USA). The column was eluted with a mixture of distilled water containing 0.1 % (v/v) trifluoroacetic acid and 0.1 % (v/v) acetonitrile. A linear gradient starting with 5 % acetonitrile was applied, reaching 20 % acetonitrile at 5 min, 30 % acetonitrile at 10 min and finally 90 % acetonitrile at 18 min. The flow rate was 2 ml.min-1 and the injection volume 20 μl. Detection was carried out with a diode array detector measuring spectra between 220 and 500 nm. The chromatograms presented are based on detection at 355 nm.

NMR measurements

IH NMR measurements were performed in D2O at 25 ⁰ C on a Varian Mercury

VX 400 MHZ NMR.

Liquid chromatography/Mass Spectrometry

LC/MS analysis was performed according to standard procedures.

Results

MonoHER is oxidized by the enzyme horseradish peroxidase

The UVAT[S spectrum of the parent compound monoHER (50 μM) is depicted in Figure 6A. Two absorption maxima are observed, one at 258 nm and another one at 355 nm (Fig. 6A). The HPLC chromatogram of monoHER recorded at 258 nm shows one major peak (tR = 6.7 min) (Fig. 7A). Addition of the enzyme horseradish peroxidase (HRP) to 50 μM monoHER changed the UVA ⁷ IS spectrum in time (Fig. 6B). Four isosbestic points are observed at 249 nm, 272 nm, 305 nm and 412 nm, suggesting the oxidation of monoHER. MonoHER consumption was confirmed by HPLC analysis (Fig. 7B). In the chromatogram, the peak height of monoHER (tR = 6.7 min) decreased after 5 minutes incubation with HRP. The average rate of monoHER oxidation at the condition applied is 13 ± 1 μM/min.

Ascorbate recycles oxidized monoHER

The UVA ⁷ IS spectrum of the solution containing monoHER with HRP in the presence of ascorbate (50 μM) is shown in Figure 6C. The spectrum of the parent compound monoHER was preserved throughout the experiment. This indicates that ascorbate prevents the accumulation of oxidized monoHER. This was confirmed by HPLC analysis (Fig. 7C). In the chromatogram, the peak height of monoHER does not decrease when monoHER is incubated with HRP in the presence of ascorbate. At the same time the ascorbate concentration decreases, as determined spectrophotometrically at 270 nm. The average rate of ascorbate oxidation is 5.15 ± 1.5 μM/min. In the absence of monoHER there was no ascorbate consumption. These data suggest that monoHER is oxidized and that the oxidation product formed is immediately regenerated by ascorbate. However, the consumption of ascorbate in the presence of monoHER (5.15 ± 1.5 μM/min) was lower than the formation of the oxidation product in the absence of ascorbate (13 ± 1 μM/min). Assuming that the oxidation product of monoHER and ascorbate react 1:1 and since no oxidation of monoHER was observed when ascorbate is present, the difference in rate between monoHER consumption in the incubation without ascorbate (13 ± 1 μM/min) and the

ascorbate consumption in the incubation with monoHER and ascorbate (5.15 ± 1.5 μM/min) is attributed to the inhibition of HRP by ascorbate. Inhibition depends on the ascorbate concentration (Figure 9). At 100 μM ascorbate, no monoHER and hardly any ascorbate was consumed indicating that HRP is almost completely inhibited.

Oxidition of monoHER in the presence of GSH leads to the formation of the 2'- monoHER-GSH adduct

The changes in the UVATtS spectrum in time after addition of 40 μM glutathione (GSH) to the incubation mixture containing monoHER and HRP are shown in Figure 6D. Four isosbestic points were observed at 265 nm, 280 nm, 311 nm and 500 nm. These isosbestic points differ from the isosbestic points observed during the oxidation of monoHER. Also, a comparison of the entire spectrum of the incubation mixtures with GSH (Fig 6D) and without GSH (Fig 6B) indicates that the products formed in both incubations are not identical. This is confirmed by HPLC analysis (Fig. 7D). In the presence of glutathione, the peak height of monoHER in the chromatogram decreases and a second peak can be detected, eluting at a position different from that of the parent compound with a retention time of 5.4 min. Based on analogy with oxidized quercetin and other oxidized phenolic compounds, it is hypothesized that this second peak represents an adduct that is formed between oxidized monoHER and GSH. The rate of this GSH adduct formation appeared to be 14.06 ± 0.03 μM/min. The rate of monoHER consumption is not affected by the presence of 40 μM GSH, indicating that GSH does not inhibit HRP. To confirm that the product formed in the presence of GSH represents a monoHER-GSH adduct and to elucidate the chemical structure of the adduct, the formed product was isolated and characterized by IH and COSY NMR. Table 4 presents the IH NMR characteristics of monoHER and the GSH adduct formed upon incubation of monoHER with HRP in the presence of GSH. The aromatic region of the IH NMR spectra of the parent compound monoHER

(Figure HA) and the GSH adduct (Figure HB) measured in D2O at 25 ⁰ C are shown in Figure 11. The doublets at 6.78 ppm and 7.37 ppm are assigned respectively to H5' and H6\ The singlets at 6.23 ppm, 6.41 ppm and 7.44 ppm are respectively from H8, H6 and H2'. These results were confirmed by the software program ACD/HNMR Predictor (v.8.09). Comparison of the IH NMR data of the GSH adduct to those of the parent compound monoHER reveals the loss of the H2' signal. Splitting patterns of all other aromatic protons are similar to those of monoHER itself. On the basis of these IH NMR characteristics it can be concluded that the GSH adduct of the oxidized flavonoid is formed in the B ring at C2'. This adduct was identified as 2'- glutathionylmonohydroxyethylrutoside (2'-monoHER-GSH adduct). The coupling of the protons was confirmed by Cosy NMR. LC/MS analysis of the purified metabolite shows an M + 1 peak at m/z 958 (data not shown). This indicates the formation of the glutathionyl adduct.

Ascorbate prevents formation of the 2'-monoHER-GSH adduct In vivo, both GSH and ascorbate are present and both compounds can react with oxidized monoHER. To determine the relative contribution of both reactions, incubations were performed containing both GSH and ascorbate. As shown in Figure 6E, addition of both GSH (40 μM) and ascorbate (40 μM) to the incubation mixture containing monoHER and HRP caused only a minor change in the UV/VIS spectrum. The rate of monoHER consumption and thus adduct formation (1.66 ± 0.30 μM/min) was slower than that in the absence of ascorbate (14.06 ± 0.03 μM). This demonstrates that the presence of ascorbate prevents GSH adduct formation. This is also deduced from the HPLC data (Figure 7E). In the chromatogram, only a minor second peak relative to the peak formed in the absence of ascorbate is formed eluting with a retention time of 5.4 min. The average rate of ascorbate consumption is 4.39 ± 1.29 μM/min.

This inhibition by ascorbate can be explained either (i) by inhibition of HRP or (ii) by competition with GSH for reaction with oxidized monoHER. As mentioned above, 40 μM ascorbate inhibits the enzyme activity by 60 %. When the rate of monoHER consumption (= GSH adduct formation) and the rate of ascorbate consumption in the presence of both 40 μM ascorbate and different concentrations of GSH (40, 500 and 5000 μM) are compared, it can be concluded that when both compounds are present in the same concentration, ascorbate reacts approximately 3 times faster than GSH with oxidized monoHER (Figure 10).

Discussion

The human body comprises an elaborate antioxidant defense system to protect cellular compounds from damage induced by free radicals, ROS and other reactive species. The different antioxidants are present at a wide range of concentrations in body fluids and tissues, with some mostly present within cells, while others are more evenly distributed throughout the body. Two of the most important endogenous hydrophilic antioxidants are glutathione (GSH) and ascorbate (vitamin C). They are present in all tissues in high intracellular concentrations ranging from 1 to 10 mM. Contradictory, their extracellular concentrations are significantly lower, i.e. approximately 1 μM GSH and 60 μM ascorbate in plasma.

During the scavenging of highly reactive species, the antioxidants donate an electron or a proton to a radical, thereby forming a relatively stable product out of the scavenged radical. However, during this reaction the antioxidant itself becomes oxidized. The formed oxidation product usually retains a part of the reactivity of the species it has scavenged and might cause damage to vital cellular targets. Therefore, the body contains a distinct network of antioxidants that can chemically reduce each other, thereby diminishing the reactivity of the formed antioxidant radical and regaining the reduced

antioxidant for the defense against reactive species. In this way, antioxidants act in synergy to destroy reactive species.

The antioxidant of our interest is the semi-synthetic flavonoid monoHER. MonoHER has been under investigation for several years as a potent protector against doxorubicin-induced cardiotoxicity because of its powerful radical scavenging and antioxidant properties.

The aim of our research was to determine how monoHER fits in the antioxidant network. Especially the interaction of oxidized monoHER between glutathione and ascorbate was investigated.

The present study shows that when monoHER exerts its antioxidant function, i.e., when it scavenges reactive species, monoHER itself is converted into a harmful oxidation product. Ascorbate recycles this oxidation product to the parent compound monoHER, while GSH reacts with it to form the 2'- monoHER-GSH adduct. The reaction with ascorbate takes place approximately 3 times faster than with GSH. In other words, when both ascorbate and GSH are present in equimolar concentration, oxidized monoHER will predominantly react with ascorbate which recycles it back to the parent compound monoHER (see Figure 12).

The implications of these findings are discussed below. Some attention will be paid to the differences and similarities between monoHER and quercetin, one of the most prominent flavonoids in our diet.

As mentioned above, a serious side effect of antioxidant reactions is the formation of harmfull oxidation products that might induce cellular damage. This phenomenon has been described for the flavonoid quercetin. Quercetin is converted into a harmful oxidation product that is prone to form adducts with

thiol groups from glutathione and proteins when it exerts its antioxidant effect.

In most cells and biofluids, that contain relatively high levels of GSH, the oxidation product of quercetin will form an adduct with the thiol GSH. In this way GSH protects against the toxic effects of the oxidation product. However, in blood plasma, where the GSH concentration is low, the oxidized antioxidant will react with other thiol groups, such as protein thiols. These protein adducts are expected to be far more stable than the GSH adduct and can therefore accumulate in the cells and cause toxicity. This protein arylation can not be prevented by ascorbate because oxidized quercetin reacts much faster with thiols than with ascorbate.

As shown in our study and in contrast to quercetin, monoHER reacts much faster with ascorbate than with glutathione. This implicates that in cells were both the GSH and ascorbate levels are high the toxic oxidation product of monoHER can be neutralized either by ascorbate or GSH. When the GSH concentration is low as seen in blood plasma, ascorbate can protect against the thiol arylation. This means that monoHER is less reactive towards thiols compared to quercetin and implicates that monoHER acts better as an antioxidant compared to quercetin.

The above mentioned differences between monoHER and quercetin can be explained by differences in their flavonoid structure. Although the chemical structures of both flavonoids are comparable, there are also some important differences. Both compounds contain a cathechol-like 3', 4'-dihydroxy substituent pattern in their B ring. This catechol moiety provides possibilities for efficient autoxidation and/or enzymatic oxidation of the flavonoid. This was demonstrated by the fact that both monoHER and quercetin become oxidized when they exert their actual antioxidant effect, leading to the formation of a harmful oxidation product. A major difference between the structure of both

flavonoids is that monoHER has a sugar group attached to position 3 of the C- ring and that the A-ring is substituted with a 7-HOCH2CH2O group. For quercetin four tautomeric forms of the oxidation product exist, one o-quinone isomer and three quinone methide isomers. GSH forms two adducts with oxidized quercetin, i.e., 6-GSQ and 8-GSQ. After monoHER oxidation, only the o-quinone isomer could be detected and only one adduct is formed in the reaction with GSH, i.e., 2'-monoHER-GSH. MonoHER lacks the C3-OH group which prevents quinone methide isomerization since the rearrangement of the proton of the 3-OH group to generate a 3-keto group can no longer occur. This restricts the structure of oxidized monoHER to the o-quinone isomer and, thus the glutathione addition to the B ring.

A large body of evidence demonstrates that monoHER can protect against oxidative stress by efficiently scavenging free radicals. During this so called protection, the antioxidant is converted into a reactive product that is converted back to its parent compound by ascorbate or captured by GSH to form a glutathionyl adduct. With this new knowledge we have to take in consideration the consequences of monoHER administration to patients receiving doxorubicin. Low intrinsic toxicity of the compound is an important requirement to allow eventual clinical application. To protect against the possible toxic effects of monoHER the ascorbate and GSH concentrations have to be high enough.

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Table 1. Patient characteristics.

Patient # Age /Sex Diagnosis Total dose of Response Biopsy dox (mg/m ² ) on dox

01 62 / F Breast cancer 100 PD N

02 54 / F Adrenal cortical cancer 150 PD N

03 25 / M Malignant peripheral 480 PR Y nerve sheet tumor 04 64 / M Malignant fibrous 450 PR Y histiocytoma 05 55 / F Breast cancer 100 PD N

06 48 / F Breast cancer 300 SD Y

07 45 / F Malignant fibrous 300 PR Y histiocytoma 08 56 / F Malignant fibrous 375 SD Y histiocytoma

Age in years; sex F female, M male; Assessment of tumor response was done by using standard ECOG criteria (PD = progressive disease; PR = partial remission; SD = stabile disease); Y yes, N no.

Table 2. Patients who received at least a cumulative dose (cum. dose) of 300 mg/m2 DOX and underwent an endomyocardial biopsy.

Patient # Biopsy score LVEF LVEF cum.dose/δt/grade before dox after > 300 mg/m ²

03 300 / 60 / Grade 3 71% 67%

04 300 / 60 / Grade 2 72% 60%

06 300 / 60 / Grade 2.5 52% ND

07 300 / 10 / Grade 3 75% 63%

08 375 / 120 / Grade 3 65% 63%

δt = time between end of monoHER infusion and start of DOX infusion in min.

The morphological grade was scored on a 6-point scale previously described by Billingham and Bristow; LVEF = left ventricular ejection fraction.

Table 3. Two patients with more than one endomyocardial biopsy.

Patient # Cum.dose / biopsy score / LVEF Biopsy score / LVEF 10 months after last cycle

03 480 / Grade 3 / 53% ND / 51 %

04 450 / Grade 2.5 / 66% Grade 2 / 62%

The morphological grade was scored on a 6-point scale previously described by Billingham and Bristow; LVEF = left ventricular ejection fraction; ND = not done.

Table 4. ¹ H NMR resonances and coupling constants of the aromatic part of monoHER and the glutathionyl adduct of monoHER.